The method for determining homocysteine in a sample and its implementation

 

(57) Abstract:

The method is intended for determining homocysteine in a sample and can be used for diagnosis and treatment in medicine and biotechnology. The sample in contact with the enzyme S-adenosylhomocysteine ( SAH-Aza) in the presence of the substrate other than homocysteine. After completion of the enzymatic reaction of the analyte appreciate without chromatographic separation of the reaction products. As the analyte using its analyte selected from adenosine analogue of adenosine and S-adenosylhomocysteine. The method does not require the use of expensive and time-consuming chromatographic separation and allows the determination in the clinical laboratory. 2 C. and 38 C.p. f-crystals.

The invention concerns a method for determining homocysteine in a sample and set for its implementation.

Homocysteine is an intermediary amino acid formed during the metabolism of methionine to cysteine. Usually homocysteine produced in the body, rapidly metabolized by one of two ways: [1] condensation with serine with the formation of citation or [2] conversion to methionine, and its concentration and the concentration of the oxidized form, homocyst in biological samples may have clinical significance in some situations, because homocysteine plays an important role in a complex series of biochemical pathways that join the metabolism containing sulfhydryl groups of amino acids, and its accumulation may indicate various violations occurring in these metabolic pathways, including, in particular, congenital abnormalities of metabolism. For example, it is known that homocystinuria (abnormal formation of homocysteine in the urine) is a violation of amino acid metabolism caused by deficiency of the enzyme citation--synthetase or the methyltransferase methyltetrahydrofolate acid, which catalyzes the methylation of homocysteine to methionine/.

Metabolism containing sulfhydryl groups of amino acids is closely associated with the metabolism of folic acid and vitamin B12/cobalamin/ that function as substrates or cofactors in various transformations. For this reason, it was also proposed to consider the accumulation of homocysteine indicator function disorders of cobalamin or folate-dependent enzymes or other disorders or diseases associated with metabolism cobalamin or folate.

In addition, since the conversion of homocysteine into methionine based on reakcje influence antifolate medicines such as methotrexate, introduced to fight with other disorders, including cancer. Therefore, it was proposed to monitor the level of homocysteine in the treatment of malignant diseases antifolate drugs.

Later it was shown that elevated levels of homocysteine in the blood correlate with the development of atherosclerosis (see Clarke, et al., New Eng. J. Med. 324: 1149-1155 /1991/, and even moderate homocysteinemia is now seen as a risk factor for heart and vascular diseases. Thus, measurement of the levels of homocysteine in plasma or blood is also of value in the diagnosis and treatment of vascular diseases.

Although immunological methods for direct determination of homocysteine is not available due to lack of available antibodies to homocysteine has been proposed a number of other methods for determining homocysteine in a clinical trial. They involve chromatographic separation and are usually based on one of the following principles:

(1) classical chromatographic analysis of amino acids

(2) the reaction of homocysteine in the sample with the enzyme S-adenosyl-L-homocysteinemia in the presence of radioactive or otherwise labeled S-adenusi-adenosyl-L-homocysteine, SAN/. Typically used chromatographic separation /IHVR or thin-layer chromatography/ and radioactivity measurement /see Refsum et al., Clin. Chem. 31:624-628 /1985/; Kredich et ai., Anal. Biochem. 116:503-510 /1981/; Chui. Am. J. Clin. Path. 90 /4/: 446-449 /1988/; Totani et al., Biochem. Soc. 14/6/:1172-9 /1988/; and Schimizu et al., Biotechnol. Appl. Biochem. 8:153 to 159 /1986//

(3) the reaction of homocysteine in the sample with a fluorophore and subsequent separation using GHWR and fluorometry /see Refsum. et al., Clin. Chem, 35 (9): 1921-1927 (1989)).

These methods are laborious, time consuming and based on direct quantification. More specifically, the chromatographic separation is a common feature of the methods used previously, and requires highly specialized and difficult-to-use equipment.

The use of such equipment is usually not welcome in routine clinical practice laboratory and therefore these methods are generally not amenable to automation in a typical clinical laboratory procedures.

There is therefore a need to develop improved test for homocysteine, which would be simple, specific, fast, adaptable for use in clinical laboratories and, most importantly, would not require roads the biscuits such a test. Thus, one aspect of this invention is the provision of a method for determining homocysteine in a sample, providing a stage of contacting the sample with converting homocysteine enzyme, for example, S-adenosylhomocysteine /SAH/-hydrolases, and at least one substrate for this enzyme, other than homocysteine, and evaluation /preferably photometric/ without chromatographic separation of the reactants or the reaction products of its analyte selected from a co-substrate for homocysteine and products of the enzymatic conversion of homocysteine above-mentioned enzyme.

After contacting the sample with converting homocysteine enzyme and its substrate is preferably incubated for at least 30 seconds, in particular at least 5 minutes before conducting subsequent stages.

As converting homocysteine enzyme used in the test of the invention, particularly preferred SAH hydrolase, but can be applied to other enzymes. Such enzymes can be, for example, betaine-homocysteinaemia and other enzymes involved in the transformation of homocysteine, as described, for example, Graham b Trends Cardiovasc. Med. 1:244 - 249 /1991//.

Co-substrate for homocysteine, the EBM in catalyzed by the enzyme for example, SAH-hydrolases, reaction conversion of homocysteine.

It is implied that used here, the term "assessment" means both quantitative and qualitative determination in the sense of obtaining an absolute value for the amount or concentration of the analyte, for example, present in the sample co-substrate for homocysteine, but also in the sense of obtaining the metric relations, percentage, visual or other value indicating about the level of the analyte in the sample. The assessment may be direct or indirect, and in fact defined chemical molecules, of course, should not be necessarily by the analyte, and can be, for example, its derivatives or any other substance that is discussed below.

In the method according to the invention are used either enzymatic or immunological methods for the estimation of the analyte. In one preferred enzymatic methods analyte is brought into contact with the enzyme for which it is a substrate, and evaluate or co-substrate, either direct or indirect reaction product of the enzymatic conversion of the analyte by this enzyme. In the preferred immunological method analyte evaluated using a procedure involving competitive svyazivayuschego or of unbound hapten.

The preferred conversion of homocysteine enzyme in accordance with this invention is S-adenosylhomocysteine /SAH hydrolase that catalyzes the reaction of adenosine + homocysteine S-adenosylhomocysteine /SAH/. The reaction has an equilibrium constant K 106M-1.

The reaction may proceed in either direction depending on the reaction conditions, reactant concentrations, etc.

In the reaction scheme adenosine is a co-substrate for homocysteine. However, in the test method of the invention can be applied to other substrates, such as analogues of adenosine or similar connection.

The invention has the special advantage that homocysteine acts as an inhibitor of SAH-hydrolases, suppressing the hydrolysis reaction, in which are formed homocysteine and adenosine, and shifting the equilibrium of the reaction in favor of the synthesis of SAH.

Thus, the amount of homocysteine in the sample indirectly affects the formation or the use of co-substrate for homocysteine, for example, adenosine, SAH-hydrolases and thus its final concentration in the reaction mixture. In the invention, the final concentration or change in concentration for the subst the th concentration of homocysteine in the sample. So, if the analyte is co-substrate, the method according to the invention differs from known methods in that instead of direct evaluation of homocysteine evaluated indirectly by determining the concentration of co-substrate when it is catalyzed by the enzyme reaction. This gives a direct benefit that can be applied to methods of detection, which is suitable for a typical clinical laboratory procedures, but cannot be applied in previous tests homocysteine, for example, photometric methods, which makes the determination of homocysteine in accordance with this invention is especially suitable for routine clinical application.

In a preferred variant of the method according to the invention, SAH-gidroliznaja reaction can be performed in any direction. So, if the test sample is in contact with adenosine and SAH-hydrolases, the amount of adenosine corresponding to the amount of homocysteine, and the amount of homocysteine in a sample can, therefore, be determined by the change in the concentration of adenosine. Instead of adenosine can be applied analogues and /or/ generiruushaya adenosine compounds.

In other preferred embodiments, it is possible to use counter is the result of hydrolysis of SAH are formed homocysteine and adenosine. Any homocysteine present in the test sample, would counteract the net reaction and, consequently, to inhibit the formation of adenosine, a number of which see.

Substrates SAH-hydrolases used in the method of the invention can therefore be SAH or adenosine or its analogues and precursors.

Many enzymes are involved in a complex series of biochemical pathways of metabolism, containing sulfhydryl groups of amino acids in the body. These paths and reactions are well studied and investigated the regulatory role of the participating enzymes. The role of one of these enzymes, SAH-hydrolases discussed in the review Ueland in Pharmacological Rewiews 34:223-253 /1982/. Trewyn et al., in J. Biochem. Biophys. Met. 4:299-307 /1981/ describe the study of the regulatory role SAH-hydrolases and offer a test for SAH-gidrolizny enzymatic activity. Carras et al., in Analytical Biochem., 199: 112 - 118 /1991/ describe other reaction pathways involving homocysteine and, in particular, provided the test MediaLounge meteosensitivity the conversion of homocysteine. Co-substrates and products of transformation of these various reactions can be applied as an analyte in the test of the present invention, in particular, the application of immunological assessment tools.

Clicou fluid or tissue extract and can be predobrabotki before testing. However, as a rule, we use the sample of blood plasma or urine sample.

In plasma or urine of a significant part of the present homocysteine may be linked by disulfide bonds with circulating proteins, such as albumin, and homocysteine may also be present in the form of other disulfide derivatives of the /usually in the form of conjugates homocysteine-cysteine/. To estimate the total amount present in the sample of homocysteine may be desirable to process the sample regenerating agent for the cleavage of disulfide bonds and free cysteine.

Disulfides easily and specifically reversed by thiol /for example, dithiothreitol /DTT/, dithioerythritol (DTE), 2-mercaptoethanol, thioglycolic acid, glutathione and similar compounds/. Direct chemical reduction can be achieved with the use of borhydride /for example, sodium borohydride/ or amalgam /for example, sodium amalgam/ or more specific reagents, such as phosphines or phosphorothioate. Recovery disulfides considered in the review by Jocelyn in Methods of Enzymology 143: 243 - 256 /1987/, which shows a wide range of suitable reducing agents.

Adenosine or dBi, based on photometric /for example, colorimetric, spectrophotometric or fluorometrically/ detection, as well as immunological methods, because they can be easily adapted for use in clinical laboratories. Particularly preferred methods based on enzymatic reactions or reactions with mono - or polyclonal antibodies, because they are simple, quick and relatively inexpensive. For example, the analyte can be estimated by monitoring the reaction with enzymes, which convert it directly or indirectly, in the products, which can be detected photometrically, for example, spectrophotometric. Appropriate enzymes, which, of course, must not react with other substrates converting homocysteine enzyme, in particular, homocysteine, are adelaideans /converting adenosine to inosine/ and adenosines /converting adenosine and ATP into ADP and phosphorylated adenosine/. Such enzymes can be combined with other enzymes, which convert the resulting products are further detected products.

Examples of immunological methods include methods involving the reaction analytie with the formation of a detectable product, for example, in a sandwich assay. However, one particularly attractive method involves the use of labeled fluorophores analogue of the analyte, preferably co-substrate, for example, labeled fluorescein adenosine, which, together with the unlabeled analyte can be brought into contact with an antibody to the analyte. If the resulting product is subjected to fluorescence polarization analysis using polarized exciting radiation, the reading of the concentration of unlabeled analyte can be made then of the degree of depolarization of the fluorescence radiation. Antibodies to adenosine commercially available /for example, Paessel & Lorei GmbH, Frankfurt, Germany and Serotech Ltd., Oxford, United Kingdom/, and fluorescence polarization immunoassay /FPIA/ well-designed /see for example, US-A-4420568 and US-A-4593089 and other publications Abbot Laboratories related to their TDX technology/.

Thus, examples of detection schemes used in the test of this invention include reaction

< / BR>
or labeled with a fluorophore of adenosine, which competes for the ATP /adenosines, with the assessment carried out by measuring fluorescence polarization.

As for schemes /2/ and /3/, inosine and uric acid have is characteristic the response.

However, the use of UV detection of uric acid or inosine has some limitations, namely that the sensitivity of the method is quite low and it requires a source of UV light and the presence of transparent for UV container for the sample. Therefore it is more convenient to rely on colorimetric detection or electronic sensors, moreover, that such methods are, in particular, colorimetric, usually preferred in clinical laboratories.

In this regard, particularly applicable reaction schema /2/ in which the generated adenozindeaminaza by the reaction of ammonia can easily detect known colorimetric methods. For example, the ammonia generated in the sample can react with the formation of colored products, the formation of which can be detected spectrophotometrically. One such method described in Methods of Enzymatic Analysis /Bergmeyer/ Volume 1: 1049-1056 /1970/ based on the reaction of ammonia with phenol in the presence of hypochlorite in alkaline conditions with formation of a colored dye Indianola

< / BR>
As a catalyst it is possible to use sodium nitroprusside. You can also use a modification of this method, for example, with the use of various derivative phenomena and therefore, adenosine in the sample.

In the scheme /3/ reaction of xanthine oxidase suitable for detection using fluorogenic or Chromogens, for example, redox indicators, by assessing the redox potential, or by measuring the consumption of O2or formation of H2O2for example, using electronic sensors. For this purpose it is possible to apply various redox indicators and the literature describes a wide range of methods for estimation of H2O2and O2in the solution. In practice, H2O2frequently detected in clinical tests.

Suitable for these purposes redox indicators are methylene blue, 2,6-dichlorphenol, indophenol and different redox indicators, the list of which is given in table 1 Kodak Research Laboratory Products Catalog N 53, although, of course, you can apply and others. To accelerate the redox reactions can be added enzymes with peroxidase activity, such as horseradish peroxidase.

If desired precipitating Chromogen, you can use the MTT tetrazolium in combination with xanthine oxidase or other similar enzymes. By applying precipitating Chromogen or fluorogenic reagent can be obtained reading immobilized the NCI concentration of adenosine is used chemiluminescent ATP reaction. Based on chemiluminescence tests have a high potential due to low limitirovany detection and relative simplicity of the required equipment. Chemiluminescent reactions can be used for detection of such analytes as ATP or H2O2and one of the most effective and the most famous of these reactions is the reaction of Firefly bioluminescence.

< / BR>
Luciferin Firefly has benzothiazoline structure, but the available luciferin from other biological sources having other structures. For analytical purposes ATP, luciferin or luciferase can be determined directly by using this reaction. You can use a different chemiluminescent reaction, which produces H2O2for example, in the layout /3/. This reaction is the reaction lyuminola /5-amino-2,3-dihydrophenazine-1,4-dione/ hydrogenperoxide catalyst, which leads to emission of light at 425 nm.

Hydrogen peroxide, for example from scheme /3/ can also be evaluated using a non-enzymatic chemiluminescent reactions of peroxyacetate and esters of acridine, the latter in aqueous solution at neutral pH.

The use of the sword is stvie relatively broad substrate specificity adenosines. In addition, this broad specificity can be used to compensate for endogenous adenosine /or other nucleoside substrates adenosines/ by adding adenosines to the sample in the form of preprocessing, preferably in combination with a regenerating agent /for example, DDT/.

Such enzymatic treatment of the sample is desirable, because in many embodiments of the invention the analyte /for example, adenosine/ already present in the sample variable supplies in quantities, providing, thus, a potential source of error in the test. Background concentration of the analyte can be compensated by carrying out a test on part of the sample without the use of converting homocysteine enzyme /for example, SAH-Gipromez/; however, this procedure takes time and makes the test more cumbersome. The alternative is the pretreatment of the sample with an agent serving to convert or remove endogenous analyte, for example, an enzyme such as adelaideans that removes background adenosine. As mentioned above, in order to avoid the need to increase the required test time such processing samples is convenient to carry out during the preprocessing of the samples regenerating agent for the release of homocysteine.

Chrome other photometric methods. Among the most applicable methods that can be applied are the agglutination of the particles and the way thus. When using polyclonal antibodies can be used direct agglutination particles or direct immunoprecipitation, although using SAH-hydrolases as converting homocysteine enzyme this is not usually preferred. However, in this case, you can use methods of inhibiting the precipitation and inhibition of agglutination of the particles. They are based on the use of combinations of antibody/hapten, leading to precipitation /deposition/ or agglutination of particles that can be detected by turbidimetric or measurement values. When the inhibition of the complex formation of antibody/hapten-analyte, for example, SAH, the content of SAH can be measured in terms of reduction of precipitation/aggregation. The reaction can be expressed as follows:

< / BR>
When running the test of the invention using SAH-hydrolases as converting homocysteine enzyme preferably either store SAH-hydrolase in the presence of a reducing agent or process its regenerating agent before use in the test. It was found that granvania during storage or causes reactivation prior to use.

You can apply various reducing reagents /for example, DTT, cysteine, mercaptoethanol, dithioerythritol, sodium borohydride, etc./, however, particularly suitable DDT, for example, at a concentration of 5 mm. Himself DTT should be stored at low pH and, therefore, the test set includes the DTT solution at low pH (for example, about 3/, but with low buffer capacity and a separate solution SAH-hydrolases, which may be partially or completely inactive at neutral pH and in the presence of buffer. When combining these solutions reactivated enzyme at neutral pH. This integration solutions can, if desired, carried out in the presence of the test sample or adding test samples soon after this, so at the same time there is a release of homocysteine. As for stabilization /activation SAH-hydrolases and to recover samples for the release of homocysteine can be applied to other above-mentioned reducing agents.

The use of reducing agents for the reactivation of inactivated SAH-hydrolases is another aspect of the present invention. Another aspect of the invention is the provision of a set, containing the first part of the inactive SAH-hydrolase, and in the second section of the manhole can be mixed with the regenerating agent and thus be re-activated immediately before use in the test.

Other additives can also be used to enhance stability SAH-hydrolases during storage and in the test. Such excipients are OVER+, glutathione, polyhydric alcohols and sugar /for example, Inositol, sorbitol, xylitol, aritra, glycerin, ethylenglycol, sucrose, lactic etc. / soluble polymers, such as some dextrans and proteins /for example, carrier proteins/.

In the case of use in the method according to the invention antibodies can be polyclonal, but are preferably monoclonal. If commercial antibodies are not available, you can get them in standard ways. Thus, antibodies can be obtained in animals or in hybridomas, either monoclonal or polyclonal, for example, as described by James Gooding in "Monoclonal antibodies, principle and practice", Academic Press, London, 1983, Chapter 3. Monoclone should be subjected to sorting for selection of clones that distinguish the target hapten among other substrates for the enzyme /enzymes/, for example, that there are adenosine and SAH. Polyclonal antibodies reactive only with the analyte /for example, SAH/ should be cleaned to remove cross-reacting antibodies, i.e. antibodies reactive with other substrates in addition to the analyte, adenosine in case if the analyte is SAH.

When antibodies are used as hapten either the analyte or another molecule containing a portion of the analyte being evaluated as the most appropriate area of the binding, for example, an area remote from the areas involved in the enzymatic reaction. The hapten anywhereman appropriately with a macromolecule, such as bovine serum albumin /BSA/ or hemocyanin. For SAH target epitope is preferably at thioester bridge or near it, and, therefore, although you can use himself SAH

< / BR>
conjugated to a macromolecule, but still preferable to use the "simplified" molecule such as a molecule of the formula I

< / BR>
where

R1and R2which may be identical or different, denote hydrogen atoms or or4groups /where R4indicates the lowest, for example, C1-6in particular, C1-6, aliphatic group such as alkyl, preferably methyl or ethyl/ or R1and R2together denote an oxygen atom, and R3represents amino or carboxypropyl/ or its salt or ester (for example, C1-4-alkanols, also connected to the macromolecule.

Examples of compounds of formula forgiven" structure can also be adopted for the above-mentioned labeled analogues, applicable to texts in which the analyte and labeled analogue are involved in the reaction of competitive binding with antibodies. So that gives the signal part of the molecule R*that can be selected from fluorophores, chromophores, radioactive labels, enzymes, chemiluminescent and other labels commonly used in immunoassay, can be conjugated with the analyte such as, for example, R*- SAH were simplified containing the epitope molecule, such as

< / BR>
Such labeled part of the molecule can be, of course, connected with particles, polymers, proteins or other materials, if desired.

Labeled 6-thioethers furanose and the compounds of formula I are novel and represent a further aspect of the present invention.

In connection with another aspect of the invention provides a method for obtaining compounds of formula I, providing at least one of the following stages:

/a/ reaction of compounds of formula II

< / BR>
in which

R1and R2have the above values, and each R5indicates a protected hydroxy-group, or both R5together denote alkylenedioxy /ie protected biomicroscopy, such as-OC(CH3)2O-SUB>3group or protected R3group, and Hal denotes a halogen atom, e.g. a bromine atom/, with the subsequent removal of any protective groups, if this is desirable;

/b/ to obtain the compounds of formula I in which R3denotes the amino group of the reaction of the compound of formula II with Acrylonitrile and recovery and removal of protective groups received cyanopropionic of tiefer;

/c/ esterification of the compounds of formula I in which R3denotes carboxypropyl.

The initial products of formula III can be obtained by standard means or known from the literature. The initial products of formula II can be obtained from the corresponding 1-hydroxymethylbilane by protecting CIS-hydroxyl groups, synthesized and subsequent reaction with thiourea and hydrolysis. CIS-hydroxy-group 1-hydroxymethylbilane can be protected by reaction with a conventional protecting the hydroxy-group agents, for example, with acetone.

Examples of schemes of reactions to produce compounds of formula I are /connections /1/ and /3/ of commercially available/

< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
< / BR>
UV-absorption, absorption of visible light or fluorescence of the substances in the reaction is ke reaction /when the signal is stable and/ or in one or more fixed time points or, alternatively, it may be kinetic dimension, in which at different points of time spend several dimensions.

For the test of the invention the necessary reagents can be added to the reaction mixture sequentially or simultaneously. However, in many preferred embodiments, one or more reactions can preferably go for some time before adding the reagents for subsequent reactions /responses/.

For example, the reaction of the test sample with adenosine and SAH-hydrolases education of SAH homocysteine and adenosine bound to happen for some time before the assessment procedure adenosine.

In clinical chemical analysis standard practice is the use of standard curves for calibration purposes. Thus, when carrying out the method of this invention can be used samples with known content of homocysteine instead of clinical samples to plot a standard curve for the measured response/signal. The homocysteine unknown samples can then be calculated by interpolation from the standard curve. Thus, precise determination of the amount forming the signal molecules or redox potentials is lugenia abnormal or potentially abnormal conditions, associated with homocysteine or appear in a certain homocysteine in the body fluids or tissues. Such conditions include atherosclerosis, blood diseases, vitamin deficiency and /or/ inborn errors of metabolism. It can be used also for evaluating the effect of pharmaceutical substances, such as antifolate medicines.

In another aspect the invention provides an analytical product, sometimes in the form of a set /China/, for use in determining homocysteine in a sample containing converting homocysteine enzyme, a substrate for this enzyme, other than homocysteine, which generates a signal agent and sometimes a means to evaluate the signal.

In one preferred options analytical product contains: converting homocysteine enzyme, for example, S-adenosylhomocysteine; one or more substrates for this enzyme, other than homocysteine; means for generating a program derived analyte selected from a co-substrate for homocysteine and products of the enzymatic conversion of homocysteine; and sometimes the means to spectrophotometric or colorimetric evaluation program production the inhabitants: adenosine; S-adenosylhomocysteine; converting enzyme adenosine; sometimes co-substrate for converting the enzyme adenosine and means for generating photometrically detected response of the co-substrate or product of the enzymatic conversion of adenosine converting enzyme adenosine. For example, converting adenosine by the enzyme can be adenosines, and means for generating a detected response may contain ATP, luciferin and luciferase. Alternatively, converting adenosine by the enzyme can be adelaideans, and means for turning may contain nucleosidases, xanthine oxidase and peroxidase.

In another embodiment, the kit contains adenosine; S-adenosylhomocysteine; sometimes associated with particles of matrix antibodies against S-adenosylhomocysteine; polegate for these antibodies, and sometimes funds for photometric evaluation of agglutination or precipitation of the complexes antibody:polegate. In this embodiment, as a convenient polioptera can be applied polymer frame, which is conjugated to a multiple 6-thioesters furanose, for example, leaving the tank remains formulas

< / BR>
They can be obtained, for example, by reaction of carboxylic KIS is th as side chains (for example, polylysin/ or amine of formula I with a polymer having side carboxypropyl.

Sets all or some of the reagents may be present in dry form, such as a matrix for processing reactions reaction medium. Similarly, the set may contain, as described above, as means for assessing the detected analyte or derivative relatively inexpensive spectrophotometric or coulometrically device, for example, the light source and the detector device for detecting the intensity of light at a wavelength characteristic of the detected analyte, etc. or even a simple coulometrically calibration chart.

Hereinafter the invention will be described using the following not limiting the invention to the examples. Particularly preferred test example 19.

Example 1. The sample /water solution homocysteine calibration or plasma or urine for clinical analysis/ preoperatively regenerating agent /for example, 10 mm dithiothreitol/. This sample was added, preferably to a final concentration of homocysteine in the range of 10-6- 10-5mol/l, the solution at 37oC containing rabbit IgG (5 mg/ml), adresinden treitel, 10 mmol/l phosphate, brought the pH to 7.4, was added 100 IU S-adenosyl-1-homocysteinemia. Simultaneously or sequentially, but preferably after 10 minutes of incubation for the purpose of conversion of adenosine within the sample, was added adenosine at a final concentration of 510-6mol/l UV-absorption was measured during 10 min and the response was measured at 292 nm kinetically, and then counted

Clinical tests the concentration of homocysteine can be calculated by interpolation on a standard curve obtained using known standards.

Example 2. Sample /like for example 1/ preoperatively regenerating agent /for example, 10 mm dithiothreitol/. This sample was added, preferably, to a final concentration of homocysteine in the range of 10-6- 10-5mol/l, the solution at 37oC containing rabbit IgG (5 mg/ml), adelaideans (20 IU/ml), xanthine oxidase (20 IU/ml), nucleosidases (20 IU/ml), horseradish peroxidase (500 IU/ml), 10 mmol/l dithiothreitol and 100 mmol/l phosphate, brought the pH to 7.4, and added 20 IU S-adenosyl-1-homocysteinemia. Then, preferably after 10 minutes of incubation to complete the conversion of adenosine in the UV over a period of 10 minutes, were calculated at 292 nm kinetic method.

Clinical tests the concentration of homocysteine can be calculated by interpolation on a standard curve obtained with known standards.

Example 3.

Test buffer:

0.1 M phosphate buffer pH of 7.4, containing 1 mg/ml rabbit IgG and 10 mmol/l dithiothreitol.

Method of test:

To 150 ml of test buffer was added to 3 IU of S-adenosyl-1-homocysteines and the sample is/ are preferably preprocessed as described in examples 1 and 2. The enzymatic reaction was started by addition of adenosine dissolved in the test buffer to a final concentration of 2,510-5mol/l After 10 min incubation was added 750 μl of a solution of test buffer containing 20 IU adelaideans, 20 IU nucleosidases, 20 IU of xanthine oxidase and 375 IU of horseradish peroxidase. The UV absorption at 292 nm was measured for 5 min kinetic method. Counted in Parallel repeated the test, but without the addition of S-adenosyl-1-homocysteinemia. Determined the difference between the two values and the concentration of homocysteine was calculated by interpolation using the standard curve.

Example 4. The test method was performed as described in example 3, including desin and monoclonal antibodies against adenosine. The remaining adenosine and labeled fluorescent adenosine compete for binding to the antibodies. The amount of bound antibody labeled adenosine assess using conventional fluorescence polarization method and the concentration of homocysteine calculated by interpolation using the standard curve.

Example 5.

Test buffer:

0.1 M phosphate buffer, of 7.4, containing 1 mg/ml rabbit IgG and 10 mmol/l dithiothreitol.

The solution SAH-hydrolases:

40 IU/ml of S-adenosyl-1-homocysteinemia dissolved in the test buffer.

A solution of adenosine:

510-8mol/ml adenosine dissolved in the test buffer.

The solution adelaideans:

200 IU/ml adelaideans dissolved in the test buffer.

The phenol solution of sodium nitroprusside:

10 mg/ml phenol, and 50 μg/ml of sodium nitroprusside dissolved in water.

Hypochlorite solution is:

11 mol/l NaOCl dissolved in 125 mm NaOH.

Method of test:

1. 75 μl of a solution of adenosine and 75 μl of solution SAH-hydrolases are mixed with the sample /preferably preprocessed as described in examples 1 - 4/ and incubated at 37oC for 10 minutes

2. Add 100 ál of solution adenosinergic and 750 μl of a solution of hypochlorite. After 30 min at 37oC measure the extinction at 628 nm. Parallel to repeat the test, but without the addition of SAH-hydrolases. Determine the difference between two magnitudes of extinction at 628 nm and the concentration of homocysteine, calculated by interpolating the difference in the standard curve obtained using known standards.

Example 6.

Getting labeled fluorophores SAH.

Prepare a solution /10 mmol/l/ SAH solution in dimethylformamide and then dilute it in phosphate buffer /100 mmol/l/ pH 7.5. To this solution add isothiocyanate fluorescein to a final concentration of 1 mmol/l After 60 minutes incubation at ambient temperature, the conjugate SAH-fluorescein cleaned using GHUR using column Chromasil C-18 at 260 nm using a gradient mixture of 25 mm ammonium acetate /pH 7.0/ methanol.

Example 7.

Antibodies against SAH.

a/ Obtaining antigen:

To a solution of 1 mmol/l SAH in phosphate buffer /25 mmol/l/ pH 7,4 containing 125 mmol/l NaCl, add bovine serum albumin to a final concentration of 5 mg/ml. To this mixture is added bis/sulfosuccinimidyl/superint to a final concentration of 1 mmol/l and the mixture is left for prohouse the amino group of adenosine, but not amino homocysteine/. The protein fraction of this solution, which also provides conjugates between BSA and SAH, isolated using gel filtration using a column Pharmacia Superose 12 containing phosphate as buffer saline as eluent.

b/ development of hybridomas:

With the described antigen receive hybridoma in accordance with the methodology described James W. Gooding b "Monoclomal antibodies: Principle and Practice", Academic Press, London 1983. Chapter 3.

c/ hybrid Selection:

/i/ Hybridoma producing antibodies against SAH, identified as follows, the IgG content of distilled liquid hybrid measured by ELISA /enzyme-linked immunosorbent assay/. Then the pooled liquid are mixed in a cuvette with buffer containing phosphate /50 mmol/l, NaCl /120 mmol/l/ pH = 7.4 and rabbit antibodies /0.1 mg/ml to a final concentration of 0.1 mcmole/l mouse IgG. Labeled with fluorescein SAH, obtained according to example 6 add up to a final concentration of 0.02 µmol/l After 10 minutes of incubation, measure the degree of polarization using spectrofluorometry, provided with a device for polarization fluorescence, by measuring A = fluorescence intensity when the polarization plane of incident light of progressence, when the plane of polarization of the incident light is perpendicular to the plane of the polarization filter used to filter the light emitted. When the wavelength of the exciting light is equal to 494 nm, and the emitted light detected at 517 nm. The degree of polarization is calculated using formula (A - B)/(A + B). The low degree of polarization indicates that the monoclonal antibody does not bind SAH.

/ii/ Of hybrids selected in accordance with /i/, producing antibodies of hybridoma, reactive relative to adenosine and homocysteine, identify the following way, monoclonal antibodies against SAH from a stand of hybrid liquid is applied on the surface of the microtiter cells as described in example 9 below.14C-adenosine /or labeled14C-homocysteine/ Amercham Ltd, UK in buffer containing phosphate /25 mmol/l NaCl /120 mmol/l and 1 mg/ml rabbit IgG, with a pH of 7.4 is added to microtiter wells. After 60 minutes incubation, the wells are washed with the same buffer /not containing, of course, adenosine or homocysteine/. High values of radioactivity, kept in cells suggests that hybridoma produce antibodies that bind to the adenosine /or homocysteine/ themselves. This is a, associated with SAH in accordance with /i/, but not associated with adenosine or homocysteine in accordance with /ii/ selected to obtain monoclonal IgG. Selected hybridoma used to obtain ascites in mice or obtaining cell cultures in vitro in accordance with customary methods. Monoclonal antibodies are then isolated from the ascites or culture media in the usual way, See. James W. Gooding "Monoclonal antibodies" Principle and practice", Academic Press, London 1983.

Example 8.

Fluorescence polarization immunoassay L-homocysteine

The enzyme solution:

Phosphate buffer /50 mmol/l with a pH of 7.4, containing 0.2 mg/ml rabbit IgG, 120 mmol/l NaCl, 10 mmol/l dithiothreitol, 10 E/l S-adenosyl-1-homocysteinemia and 0.1 mmol/l adenosine.

Tagged fluorescein solution SAH:

SAH, conjugated with fluorescein obtained in accordance with example 6, was dissolved to a final concentration of 1 µmol/l in phosphate buffer /50 mmol/l/ pH 7,4 with NaCl /125 mmol/l and 0.2 mg/ml rabbit IgG.

The antibody solution:

Monoclonal antibodies against SAH /not reactive relative to adenosine and preferably in relation to homocysteine/, for example, obtained according to example 7, is dissolved to a final con is 2">

The test:

In a ditch 15 μl plasma /original series of samples with known homocysteine/ mixed with 100 μl of an enzyme solution and incubated at 37oC for 15 minutes Add 100 ál of a solution of fluorescein labeled SAH followed by the addition of 1.0 ml of a solution of antibody. Using spectrofluorometer, equipped with a device for fluorescence polarization to measure the degree of polarization, as described in example /c/ /i/ and build the curve of the dependence of the polarization on the concentration of homocysteine. The test can also be performed using labeled haptens and antibodies of the examples 11 and 13, or 15 and 17 suggested in examples 6 and 7.

Example 9.

Microtiter enzyme-linked immunosorbent assay.

/a/ Apply a solution of the enzyme of example 8.

/b/ is the Solution of peroxidase labeled SAH:

0.5 mg of horseradish peroxidase dissolved in 1 ml of purified water. 200 μl of a solution of 0.02 mol/l of periodate sodium added, the mixture is stirred for 20 minutes at ambient temperature and cialiswhat overnight against 10 mm buffer, prepared from sodium acetate with a pH of 4.4. Add SAH to a final concentration of 0.1 mmole/l and the pH was adjusted to 6.0. The solution is stirred 4 h at ambient temperature, spri 4oC for 2 hours Peroxidase and its conjugates with SAH allocate using gel filtration on a column of Superose 6 /Pharmacia, Sweden/.

/c/ Antibodies against SAH deposited on a microtiter cell:

Polyclonal sheep IgG, obtained from sheep immunized with mouse IgG, diluted to a final concentration of 1 mg/ml in borate buffer /100 mmol/l/ pH to 9.0. 300 μl of this solution make in each cell polystyrene microtiter plates. After 120 minutes incubation at 37oC cells are washed 5 times containing the phosphate salt solution. After that, mouse monoclonal IgG antibodies against SAH, obtained according to example 7, is dissolved in containing phosphate saline to a final concentration of 50 μg/ml 200 µl of monoklonalnyh IgG is added to each cell and incubated for 120 minutes at 37oC. Then cells are washed 5 times containing the phosphate salt solution containing 0.1 mg/ml rabbit IgG.

/d/ a test:

25 ál of plasma samples /first series of samples with known concentrations of homocysteine/ mixed with 500 μl of an enzyme solution and incubated at 37oC for 15 minutes Add 50 ál of a solution of peroxidase labeled SAH and after mixing 250 ál of this mixture add the After 60 min incubation at 37oC cells are washed 3 times with phosphate buffered saline containing 0.1 mg/ml rabbit IgG. 100 μl of a solution of o-phenylenediamine /1 mg/ml in citrate buffer /0.1 mol/l with a pH of 6.0, containing 0.015% hydrogen peroxide, is added to each cell. After 10 to 30 minutes to measure the absorption of light at 450 nm of each cell. Build the curve of dependence of the absorption on the concentration of homocysteine.

Example 10.

Getting hapten.

3-S-(1-anhydrous-D-ribofuranosyl)-dipropylamine

< / BR>
/a/ Activated hydroxyamine mercaptan /Connection /8/ schema /E/ above/.

< / BR>
One equivalent of methylribonucleotide /Connection /1/ above, which is commercially available/ reacts with a mixture of 5 equivalents trimethylsilylmethylamine and 1 equivalent epirate trattoria boron according to the method Jun et al. /Carb. Res. 163: 247 - 261 /1987///. 0,5 equivalent product 1 anhydride-D-ribose /Connection 2/ in finely divided form in small portions with continuous stirring, to a mixture of acetone/sulfuric acid obtained by slow addition of 6.3 ml of concentrated sulfuric acid to 100 ml of fresh acetone in a bath with ice. Bath ice is removed and the reaction is carried out at an ambient temperature of the house of sodium, dilute hydrochloric acid and in the end with water, dried and evaporated, obtaining the derivative of 2,3-isopropylidene-D-ribnovo acid 1-anhydrous-D-ribose /Connection 2/. One equivalent of this compound and two equivalent tetrabromide carbon dissolved in dry ether and cooled on ice. With constant stirring and cooling in an ice slowly add two equivalents of triphenylphosphine. Bath ice is removed, allowing the mixture to warm to ambient temperature at which the reaction begins and evenly allocated hydrogen bromide. After completion of the reaction, excess reagent is quenched by adding methanol. Bromine derived /Connection /6// allocate by filtration and evaporation of the filtrate. To breedname derived add one equivalent of thiourea dissolved in warm water and diluted with purified alcohol. The mixture is refluxed and periodically shake well, continuing this operation approximately 30 minutes after dilution bromides derived. The reaction mixture is cooled on ice and filtered, obtaining a solid substance, which is treated with alkaline water with the formation of hydroxyamino mercaptan /Connection /7// in the organic phase. His turn in advance is described below.

/b/ 3-S-(1-anhydrous-D-ribofuranosyl)dipropylamine

One equivalent of freshly prepared compound of example 10 /a/ /Connection /8// reacts with one equivalent of Acrylonitrile with the formation of tiefer /Connection /9//. Then restore processing LiAIH4in dry ether and free unprotected amine /Connection /10// allocate processing aqueous hydrochloric acid. Appropriate aminopropylene esters in which R1and R2other than hydrogen, get similarly, for example, using commercially available compounds /1/ and /3/ as the source materials.

Example 11.

The tagging of the hapten.

A solution of 50 mmol/l compound of example 10 in dimethylformamide /DMF/ diluted 1:5 /volume/ 0.1 M solution of bicarbonate /pH of 9.2/. To this solution add isothiocyanate fluorescein to a final concentration of 12 mmol/l After 60 minutes incubation at ambient temperature fluorescein conjugate with the compound of example 10 purify by chromatography with reversed phase /RPC/ using Kromasil column and a gradient of a mixture of 20 mm ammonium acetate /pH 7.0/ methanol.

Example 11.

Obtaining antigen.

To a solution of 1 mmol/l of the compound of example 10 in phosphate and 5 mg/ml To this mixture is added bis(sulfosuccinimidyl)superint to a final concentration of 1.2 mmol/l and the mixture is left to react for 60 minutes the Protein fraction containing conjugate the hapten-BSA, isolated using gel filtration using a column Pharmacia Superose 12 containing the phosphate salt solution as eluant.

Example 13.

Obtaining antibodies.

Antibodies to the compound of example 10 get analogously to example 7, using the antigen of example 12. Antibodies that do not react with SAH, and antibodies reactive adenosine, drop, and preferably antibodies reactive against homocysteine.

Example 14.

Getting hapten.

N-hydroxysuccinimidyl-C-S-(1-anhydrous-D-ribofuranosyl)thio - butanoate

< / BR>
One equivalent of the compound of example 10 /a/, freshly prepared, reacts with 1 equivalent of ethyl-4-bromobutyrate with the formation of the essential connection /11/. It hydrolyzing free acid by means of alkaline hydrolysis of aqueous sodium hydroxide in dioxane and remove the protective group by treatment of aqueous hydrochloric acid with the formation of unprotected free acid /Connection /12//. One equivalent of the compound sment dicyclohexylcarbodiimide with constant stirring. The reaction proceeds at ambient temperature for 18 hours, after which the mixture cool and add chilled on ice ether. Beleaguered NHS-ester /Connection /13// recrystallized from a mixture of dimethylformamide with ether, dried and then stored at 4oC over dessicant.

Relevant NHS-esters in which R1and R2other than hydrogen, get similarly, for example using commercially available compounds /1/ and /3/ as the source materials.

Example 15.

The tagging of the hapten.

A solution of 50 mmol/l compound of example 14 in dimethylformamide //DMF/ diluted 1:5 /volume/ in 0.1 M bicarbonate buffer /pH of 9.2/. To this solution was added 5-aminoacetanilide /amide of fluorescencia/ a final concentration of 12 mmol/l After 60 min incubation at ambient temperature fluorescein conjugate with 3-S-(1-anhydrous-D-ribofuranosyl)-timesnow acid was purified by chromatography with reversed phase using column Kromasil C-18, gradient a mixture of 20 mm ammonium acetate and methanol.

Example 16.

Obtaining antigen.

To a solution of BSA (5 mg/l in phosphate buffer (50 mmol/l) containing 125 mmol/l NaCl, pH 7,4// add connection note the containing conjugate BSA-hapten, allocate using gel filtration using a column Pharmacia Superose 12 containing the phosphate salt solution as eluant.

Example 17.

Obtaining antibodies.

Antibodies to the compound of example 14 receive analogously to example 7 with the use of an antigen of example 12. Antibodies that do not react with SAH, and antibodies reactive with adenosine, was discarded as antibodies reactive against homocysteine.

Example 18.

Fluorescence polarization immunoassay

The enzyme solution:

50 mmol/l phosphate buffer /pH 7,4/ containing 4 mg/ml casein, 120 mmol NaCl and 10 E/l S-adenosyl-L-homocysteinemia.

The solution dithiothreitol /DDT/:

Dithiothreitol dissolved in water to a concentration of 50 mmol/l and pH adjusted to 3.0 with hydrochloric acid.

A solution of adenosine:

1.8 mmol/l adenosine 50 mmol/l phosphate buffer /pH 7,4/.

A solution of fluorescein labeled SAH:

50 mmol/l phosphate buffer /pH 7,4/ containing SAH, conjugated with fluorescein formed according to example 6.

The antibody solution:

Monoclonal antibodies against SAH /for example, in accordance with example 7/ dissolved to a final concentration of 0.1 μmol/l in fo the>/P>Stage 1:

In a ditch 15 ál of sample, 10 μl of an enzyme solution and 10 μl of a solution of adenosine mixed with 10 μl of an acidic solution of DTT and incubated at 37oC turning 15 minutes

Stage 2:

To the cuvette was added 100 μl of a solution of fluorescein labeled SAH and 1.0 ml of a solution of antibody. Using spectrofluorometry, provided with a device for fluorescence polarization to measure the degree of polarization, as described in example 7 /c/ above and build a curve according to the degree of polarization depends on the concentration of homocysteine.

Example 19.

Fluorescence polarization immunoassay.

The enzyme solution:

Phosphate buffer /50 mmol/l with a pH of 7.4, containing 1 mg/ml casein, 120 mmol/l NaCl and 10 E/l S-adenosyl-L-homocysteinemia.

The solution dithiothreitol /DDT/:

Dithiothreitol dissolved in water to a concentration of 50 mmol/l and the pH was adjusted to 3.0 with hydrochloric acid.

A solution of fluorescein labeled SAH /solution of adenosine:

Phosphate buffer /50 mmol/l with a pH of 7.4, containing 10 mcmole/l conjugated to fluorescein SAH, obtained according to example 6, and 1.8 mmol/l adenosine.

The antibody solution:

Monoclonal antibodies against SAH /for example, in accordance with the use of the/l NaCl and 1 mg/ml casein.

Conducting the test.

In the cell 10 ál of plasma, 100 μl of an enzyme solution and 10 μl of a solution of labeled SAH adenosine mixed with 30 μl of an acidic solution of DDT and incubated at 37oC within 15 minutes After incubation, add 1.0 ml of a solution of antibody. Using spectrofluorometry, provided with a device for fluorescence polarization to measure the degree of polarization, as described in example 7/c/ /i/ and build the curve according to the degree of polarization depends on the concentration of homocysteine.

The tests of examples 18 and 19 can also be performed using labeled haptens and antibodies of examples 11 and 13, or 15 and 17, instead of presented in examples 6 and 7.

Example 20.

Fluorescent test.

Test buffer I:

50 mm Pipes buffer /pH 6,6/, containing 1 mg/ml casein, 10 mm DTT, 0.5 mm MgCl2and 30 mm KCl.

Test buffer II:

40 mm Hepes-buffer /pH of 7.75/ containing 4 mmol/l EDTA, 20 mm MgCl2and 0.36 mmol/l DTT.

Test buffer III:

40 mm Hepes-buffer /pH 6.75 in/ containing 1,6 μg/ml luciferase from Photinus pyralis/, 700 mmol/l D-luciferin, 20 mmol/l MgCl2, 4 mmol/l EDTA, 0.36 mmol/l DTT, and 0.3 mmol/l AMP.

Conducting the test.

To 130 μl of test buffer 1 add 3 E S-adenosyl-1-homocysteinemia, 20 intelligent in the test buffer 1, to a final concentration of 510-6mmol/l After 5 min incubation at 37oC add 750 μl of test buffer 1 containing 0,710-5mmol/l ATP, and 1 IU adenosines and the resulting solution was then incubated at 37°oC 5 minutes, This solution was diluted 1: 100 /volume/ test-buffer II and 500 µl of the diluted solution was immediately added to 500 μl of test buffer III. Both buffer II and III to balance the ambient temperature /21oC/. The luminescence read in a spectrophotometer at 550 nm.

In parallel, the test is carried out without S-adenosyl-1-homocysteinemia. Clinical tests of the concentration of homocysteine can be calculated by interpolation into a standard curve of the difference in luminescence produced in the presence of S-adenosyl-L-homocysteinemia without it.

Example 21.

Obtaining polyclonal antibodies.

Rabbit polyclonal antibodies to antigens of examples 12 and 16 received according to the Protocol Dako Corporation, Copenhagen, Denmark. Polyclonal IgG purified from collected antisera according to the same Protocol. Polyclonal antibodies purified from antibodies reactive with the remnants of adenosine and homocysteine perse, the passage of antibodies through the column Racti-Gel with immobilized residues. Sobranie antibodies can be used in the tests described above.

1. The method for determining homocysteine in a sample, comprising contacting the sample with the enzyme S-adenosylhomocysteine (SAH-Aza) in the presence of the substrate other than homocysteine, and evaluation of the analyte, characterized in that as the analyte using its analyte selected from adenosine analogue of adenosine and S-adenosylhomocysteine (SAH), and evaluate analytical methods, other than chromatographic.

2. The method according to p. 1, wherein the sample after completion of the enzymatic reaction with SAH-hydrolases contact with the antibody to the analyte in the presence of the hapten for antibody other than its analyte, in this case, the estimation of the analyte carried out according to the number of hapten bound or unbound to the antibody.

3. The method according to p. 2, wherein the hapten is polegate.

4. The method according to p. 2, wherein the hapten is a labeled molecule having a structural epitope unit, similar epitope structural unit of unlabeled analyte.

5. The method according to any of paragraphs. 2 to 4, characterized in that the antibody is MONOSEM matrix.

7. The method according to p. 1, wherein the sample after completion of the enzymatic reaction involving SAH-hydrolases in contact with a second enzyme that converts the specified analyte, in this case, the evaluation of the specified analyte carried out according to the number of the second substrate of the enzyme other than the analyte or the formed product of the transformation of the analyte.

8. The method according to p. 1 or 7, characterized in that the analyte is an adenosine.

9. The method according to p. 7, characterized in that adenosinergic the enzyme is adenosines.

10. The method according to p. 7, wherein adenosine is subjected to enzymatic conversion in the presence of adenosines and ATF for at least 1 min, then add luciferin and luciferase and register the generated light.

11. The method according to p. 7, characterized in that adenosinergic enzyme is adenoidectomies.

12. The method according to p. 7, characterized in that the adenosine estimate in the presence of adenozindezaminazy, nucleosidases, xanthine oxidase and peroxidase by the absorption of the UV.

13. The method according to any of paragraphs. 1-7, characterized in that the analyte predstavlyaet a S-adenosylhomocysteine.

15. The method according to p. 1, wherein the sample after completion of the enzymatic reaction involving SAH-hydrolases in contact with the labeled fluorophore SAH or labeled with the fluorophore 6-tieferen furanose, then add the monoclonal antibody to SAH, with a bound or unbound to the antibody labeled with the fluorophore compound judged by fluorescence polarization.

16. The method according to p. 1, wherein the sample after completion of the enzymatic reaction involving SAH-hydrolases contact with a labeled SAH or labeled 6-tieferen furanose, then add the monoclonal antibody to SAH associated with matrix media matrix media washed with antibodies associated with antibodies labeled compound evaluated photometrically.

17. The method according to p. 16, characterized in that the label associated with the antibody labelled compound is subjected to an enzymatic reaction for generating chromophore and assessed photometrically.

18. The method according to p. 3, characterized in that the evaluation carried out by precipitation or agglutination of conjugates of the antibody-polegate.

19. The method according to p. 2, characterized in that as the hapten used connect the hydrogen atoms or or4group, or together an oxygen atom or different and denote a hydrogen atom and OR4, R4represents C1-6-aliphatic group, and R3-amino - or carboxypropyl.

20. The method according to p. 4, characterized in that as the use of hapten-labeled 6-thioether furanose, while labeled part is a chromophore or fluorophore, or a radioactive atom, thioester part represents trimethylindium.

21. The method according to p. 20, characterized in that as labeled tiefer used as a compound of formula I under item 19.

22. The method according to p. 8, characterized in that SAH-hydrolase used in inactivated form, thus activating spend regenerating agent.

23. The method according to p. 1, wherein the sample is a blood sample, plasma or urine, pre-treated regenerating agent.

24. The method according to p. 1, characterized in that the estimate of the analyte is carried out photometrically.

25. The method according to p. 24, characterized in that the evaluation carried out by spectrophotometric or colorimetrically.

26. The method according to p. 24, wherein the assessment is made turbidimetric or values.

28. Analytical kit for determination of homocysteine in the sample under item 1, including SAH-ABC and the substrate, characterized in that it further contains an agent that forms a signal, and as a substrate adenosine or an analogue of adenosine, or S - adenosylhomocysteine.

29. Set on p. 28, characterized in that it further comprises means for estimating the signal.

30. Set on p. 28, characterized in that as agent for forming the signal contains a labeled SAH and do not necessarily associated with the matrix carrier antibody to SAH.

31. Set under item 29 or 30, characterized in that as a means for estimating signal includes means for photometric evaluation labeled SAH or a subclass thereof.

32. Set on p. 28, characterized in that the substrate contains adenosine.

33. Set on p. 32, characterized in that it further comprises associated with the matrix antibody to SAH and polegate for antibodies to SAH.

34. Set according to any one of paragraphs. 29 to 32, characterized in that the means for estimating signal represents a tool for photometric determination of agglutination or precipitation of the complexes of the antibody-polegate.

35. Set on p. 29, characterized in that as substi, and means for estimating a signal is a means for generating a photometrically detectable response.

36. Set on p. 35, characterized in that it further contains a co-substrate to adenosintriphosphate the enzyme.

37. Set on p. 35, wherein the enzyme is adenosines, and means for generating the response contains ATP, luciferin and luciferase.

38. Set on p. 35, wherein the enzyme is adenoidectomies, and means for generating the response contains nucleosidases, xanthine oxidase and peroxidase.

39. Set on p. 28, characterized in that it contains inactive SAH-ABC, and as activator revitalizing agent.

40. Set on p. 39, characterized in that it contains dithiothreitol in an acidic environment.

Priority points:

22.01.92 on PP. 1, 7, 8, 11, 12, 13, 23, 24, 25, 27, 28, 29;

10.02.92 on PP. 30, 31;

22.01.93 on PP. 32 - 40.

 

Same patents:
The invention relates to medicine and is intended for the detection of metastasis of lung cancer and tumors of other organs in the mediastinum
The invention relates to immunobiotechnology and can be used in the production of a highly sensitive test systems for (qualitative and quantitative) determination of antigens, antibodies and other immunoreactive compounds, as well as in the technology of beinstantly and reliable diagnostics for genetic hybridization and ligand-receptor analysis

The invention relates to medicine, namely cardiology, and can be used to diagnose early (preclinical) heart failure in patients with hypertrophic cardiomyopathy

The invention relates to medicine, specifically to methods for radionuclide diagnosis of osteomyelitis
The invention relates to medicine, in particular, tuberculosis, and can be used in anti-TB and cancer institutions

The invention relates to medicine, namely to clinical immunology, and can be used for the diagnosis of severe asthma

The invention relates to medicine, namely to immunology
The invention relates to veterinary medicine, in particular for in vivo monitoring for the presence of the pathological process in breeding birds typical of Marek's disease (BM)

The invention relates to techniques for laboratory studies, in particular to the dot enzyme-linked immunosorbent analysis intended for laboratory serological diagnosis of viral diseases, including distemper

The invention relates to medicine, namely to neuropathology, and immunology, and is intended for determining the activity of the pathological process in patients with multiple sclerosis (MS), t

The invention relates to medicine, namely to biochemical research in Microbiology, and can be used in scientific research and practical medicine for the study of mycobacterial enzymes

The invention relates to medicine, namely to ophthalmology, and relates to methods for diagnosing keratoconus

The invention relates to Enzymology, food industry and medicine and can be used to control preparations of collagenase

FIELD: chemistry.

SUBSTANCE: invention relates to a method for determination and quantitative estimation of unusually modified glycans, which can be used in analysing glycans. The disclosed method includes the following steps: a) providing a glycan preparation containing unusually modified glycans selected from a group containing sulphated glycans, phosphorylated glycans, polyacetylated sialylated glycans and combinations thereof, where said unusually modified glycans are negatively charged and where said glycan preparation is obtained by separating glycans and sialic acids from a therapeutic glycoprotein composition, where the sialic acids are separated by exposing the therapeutic glycoprotein composition to at least one agent which splits sialic acid residues in conditions which facilitate splitting of sialic acids; b) subjecting the glycan preparation to a chromatographic separation method which separates glycans based on the charge-to-mass ratio, thereby separating said unusually modified glycans; and c) quantitative determination of at least one separated unusually modified glycan using at least one quantitative estimation standard.

EFFECT: novel efficient method which enables to analyse unusually modified glycans.

42 cl, 7 dwg, 2 tbl, 1 ex

Up!